1. Field of the Invention
This invention resides in the field of plasmid DNA purification, and particularly the use of hydroxyapatite column chromatography to purify plasmid DNA.
2. Description of the Prior Art
Gene therapy is one of the most promising developments in the treatment of disease and genetic defects and disorders. Gene therapy is the introduction of a gene fragment into the cells of a patient, the fragment being one that when expressed provides a therapeutic benefit to the patient. One example of such a benefit is the expression of a protein that is otherwise not sufficiently expressed by the patient due to a defect in a native gene. Another is the blocking of an undesired genetic function, such as a viral infection or cancer, by introducing a mutant gene, an antisense sequence or a ribozyme.
One method of introducing genes is by the use of plasmid DNA as a vector, and the interest in gene therapy has generated a demand for highly purified plasmid DNA. The purification of plasmid DNA requires the complete removal of other cell components such as host proteins, endotoxins, chromosomal DNA and RNA. This is commonly achieved by culturing host cells, typically bacterial cells, that contain the plasmid DNA, recovering the cells by centrifugation, resuspending them in a suitable suspension buffer and performing cell lysis with an alkali lysing agent, then lowering the pH of the lysate, commonly with potassium acetate buffer at pH 5.5, to precipitate out chromosomal DNA and other cellular materials while leaving the plasmid DNA in the supernatant. The supernatant is then recovered and clarified, and plasmid DNA is isolated from the supernatant by chromatography. Suitable chromatographic techniques are anion exchange chromatography, size exclusion chromatography, and reverse phase high-performance liquid chromatography.
Hydroxyapatite is a particularly effective separation medium for proteins and nucleic acids, operating by mixed-mode ion exchange due to its inclusion of both positively and negatively charged moieties. Hydroxyapatite is thus known for its use in purifying plasmid DNA. The retention of acetate ion from the neutralization step however limits the effectiveness of hydroxyapatite, since the acetate ion tends to interact with the hydroxyapatite causing dissolution of the medium. To avoid this problem, the supernatant is commonly treated by precipitation, desalting, diafiltration or dialysis prior to chromatography. These steps, of course, are time-consuming, add to the cost of producing purified plasmid DNA, and may cause sample loss and/or denaturation.
It has now been discovered that when an alkaline cell lysate containing plasmid DNA is acidified by a mineral acid in the presence of an inorganic salt, rather than by the acetate buffer of the prior art, and the supernatant is removed from the resulting precipitate and applied directly to a hydroxyapatite chromatographic separation medium, purified plasmid DNA can be eluted from the separation medium. The intervening steps of treating the lysate according to the prior art can be eliminated with no loss of product quality. In addition to the cost and time advantage of eliminating these steps, increased product yield is anticipated. This discovery avoids the use of acetate ion and its degradative effect on hydroxyapatite. The hydroxyapatite is therefore available for reuse in a large number of purifications of further lysate supernatants. The present invention therefore resides in a method of extracting plasmid DNA from bacterial cells, in which a cell lysate is first formed by the action of an alkaline lysis agent, the lysate is then acidified with a mixture of a mineral acid and an inorganic salt, the resulting supernatant and precipitate are separated, and the supernatant is directly applied to the hydroxyapatite, the plasmid DNA then being eluted with an appropriate elution buffer. Further features of the invention and its preferred embodiments will be apparent from the description that follows.